1. Technical Field
The present invention is directed to nucleic acid and amino acid sequences and constructs, and methods related thereto.
2. Background
Through the development of plant genetic engineering techniques, it is now possible to produce a transgenic variety of plant species to provide plants which have novel and desirable characteristics. For example, it is now possible to genetically engineer plants for tolerance to environmental stresses, such as resistance to pathogens and tolerance to herbicides and to improve the quality characteristics of the plant, for example improved fatty acid compositions. However, the number of useful nucleotide sequences for the engineering of such characteristics is thus far limited and the speed with which new useful nucleotide sequences for engineering new characteristics is slow.
There is a need for improved means to obtain or manipulate compositions of sterols and fatty acids from biosynthetic or natural plant sources. For example, novel oil products, improved sources of synthetic triacylglycerols (triglycerides), alternative sources of commercial oils, such as tropical oils (i.e., palm kernel and coconut oils), and plant oils found in trace amounts from natural sources are desired for a variety of industrial and food uses. Or, the ability to increase sterol production in plants may provide for novel sources of sterols for use in human and animal nutrition.
To this end, the triacylglycerol (TAG) biosynthesis system and sterol biosynthesis in mammalian tissues, yeast and plants has been studied.
Sterol biosynthesis branches from the farnesyl diphosphate intermediate in the isoprenoid pathway. Sterol biosynthesis occurs via a mevalonate dependent pathway in mammals and higher plants (Goodwin,(1981) Biosynthesis of Isoprenoid Compounds, vol 1 (Porter, J. W. and Spurgeon, S. L., eds) pp.443-480, John Wiley and Sons, New York), while in green algae sterol biosynthesis is thought to occur via a mevalonate independent pathway (Schwender, et al. (1997) Physiology, Biochemistry, and Molecular Biology of Plant Lipids, (Williams, J. P., Khan, M. U., and Lem, N. W., eds) pp. 180-182, Kluwer Academic Publishers, Norwell, Mass.).
The solubility characteristics of steroyl esters suggests that this is the storage form of sterols (Chang, et al., (1997) Annu. Rev. Biochem., 66:613-638). Sterol O-acyltransferase enzymes such as acyl CoA:cholesterol acyltransferase (ACAT) catalyze the formation of cholesterol esters, and thus are key to controlling the intracellular cholesterol storage.
Such ACATs have been the subject of many research efforts, particularly for applications involving the reduction of cellular cholesterol storage in humans. Several studies suggest that cholesterol esters contribute significantly to the early formation of foam cells in atherosclerosis in humans (Fowler, et al. (1979) Lab. Invest. 41:372-378; Schaffner et al. (1980) Am. J. Pathol. 100:57-80; Lupu, et al. (1987) Arterosclerosis 67:127-142; Brown et al. (1983) Annu. Rev. Biochem. 52:223-261; the entirety of which are incorporated herein by reference) and by blocking ACAT, intracellular cholesterol esters are significantly reduced (Ross, et al. (1986) J. Biol. Chem. 259:815-819; Tabas, et al. (1986) J. Biol. Chem. 261:3147-3155; Cadigan, et al. (1988) J. Lipid Res. 29:1683-1692; Bocan et al. (1991) Arterioscler. Thromb. 11:1830-1843, the entirety of which are incorporated herein by reference). Thus, directly inhibiting ACAT within the arterial wall may inhibit the progression of atherosclerotic lesions without lowering total plasma cholesterol.
TAG biosynthesis occurs in the cytoplasmic membranes of plant seed tissues which accumulate storage triglycerides (xe2x80x9coilxe2x80x9d), fatty acyl groups are added sequentially by specific acyltransferase enzymes to the sn-1, sn-2 and sn-3 positions of glycerol-3-phosphate (G3P) to form TAG. This pathway is commonly referred to as the Kennedy or G3P pathway.
The first step in TAG formation is the acylation of the sn-1 position of glycerol-3-phosphate, catalyzed by glycerophosphate acyltransferase, to form lysophosphatidic acid. The lysophosphatidic acid is subsequently acylated at the sn-2 position by lysophosphatidic acid acyltransferase (LPAAT) to create phosphatidic acid. The phosphatidic acid is subsequently dephosphorylated at the sn-3 position by phosphatidic acid phosphatase to form sn-1,2-diacylglycerol (DAG).
An important step in the formation of TAG is the acylation of the sn-3 position of sn-1,2-diacylglycerol by diacylglycerol acyltransferase (DAGAT, EC 2.3.1.20) ultimately forming triacylglycerol (TAG).
The characterization of diacylglycerol acyltransferase (also known as DAGAT) and acyl CoA:cholesterol acyltransferase (also known as ACAT) is useful for the further study of plant fatty acid and sterol synthesis systems and for the development of novel and/or alternative sterol and oils sources. Furthermore, identification of novel ACAT sequences may provide a novel means to inhibit intracellular cholesterol ester formation in animals, thus reducing atherosclerosis. Studies of plant mechanisms may provide means to further enhance, control, modify, or otherwise alter the total fatty acyl composition of triglycerides and oils. Furthermore, the elucidation of the factor(s) critical to the natural production of triglycerides in plants is desired, including the purification of such factors and the characterization of element(s) and/or cofactors which enhance the efficiency of the system. Of particular interest are the nucleic acid sequences of genes encoding proteins which may be useful for applications in genetic engineering.
The present invention is directed to acyl-CoA:cholesterol acyltansferase (EC 2.3.1.26, also referred to herein as ACAT) related polynucleotides, and in particular to ACAT-related polynucleotides. The polynucleotides of the present invention include those derived from plant, fungal, mammalian and nematode sources.
Thus, one aspect of the present invention relates to isolated polynucleotide sequences encoding ACAT related proteins. In particular, isolated nucleic acid sequences encoding ACAT related proteins from human, rat, C. elegans, Arabidopsis, soybean and corn are provided.
Another aspect of the present invention relates to oligonucleotides which include partial or complete ACAT encoding sequences.
It is also an aspect of the present invention to provide recombinant DNA constructs which can be used for transcription or transcription and translation (expression) of ACAT. In particular, constructs are provided which are capable of transcription or transcription and translation in host cells.
In another aspect of the present invention, methods are provided for production of ACAT in a host cell or progeny thereof. In particular, host cells are transformed or transfected with a DNA construct which can be used for transcription or transcription and translation of ACAT. The recombinant cells which contain ACAT are also part of the present invention.
In a further aspect, the present invention relates to methods of using polynucleotide and polypeptide sequences to modify the sterol, sterol ester, and fatty acid content as well as composition, particularly in seed tissue of oilseed crops. Plant cells having such a modified sterol and fatty acid content are also contemplated herein.
In yet a further aspect, the present invention relates to methods of using polynucleotide and polypeptide sequences to inhibit or delay the germination of seeds.
The modified plants, seeds and oils obtained by the expression of the ACAT-like proteins are also considered part of the invention.